Osteoporosis is a common disease among the elderly, especially in postmenopausal women. Current treatments for osteoporosis are aimed at inhibiting bone resorption. Thus, treatments that promote bone formation could provide a major improvement in the therapy of osteoporosis. Bone morphorgenetic proteins (BMPs) are able to induce ectopic bone formation and have been proposed as possible agents to stimulate new bone formation in patients with osteoporosis. Downstream components of the BMP signaling pathway may also serve as targets for therapy in osteoporosis, but little is known about how BMPs signal from the cytoplasm to the nucleus, particularly how they activate gene expression in responsive cells. Our long term goals are to use the molecules we identify in Xenopus to characterize BMP regulated gene expression in other BMP responsive tissues, particularly osteogenic precusors, and to explore these molecules as potential therapeutic targets in the treatment of osteoporosis. Xenopus embryos will be used for much of the experiments described here, since they have provided a highly successful model system for the study of other BMP functions as well as the characterization of activin/TGF-beta regulated gene expression. The specific goals of this proposal are to determine the promoter sequences that mediate Mix.2 gene expression in response to BMP-4 in Xenopus laevis and to characterize the transcription factors that interact with these sequences. Our preliminary evidence suggests a direct role for Smad proteins in the induction of the Mix.2 gene, and this role will be further explored by characterizing the interactions between Smad genes, BMP-4 regulated transcription factors, and the DNA elements within the BMP-4 regulated promoter. Several novel genes have been identified in a yeast two-hybrid screen that interact with the Smad1 protein. These clones will be characterized further by examining their patterns of expression and their ability to interact in vivo with Smad1 and/or transcription factor complexes regulating Mix.2 gene expression. In future studies, these proteins characterized from Xenopus will be identified in mammalian tissues, particularly osteogenic precursor cells, and examined for their roles in mediating bone formation in mammals in response to BMPs.